The Rho family of p21 small GTPases plays important roles in regulating cytoskeleton rearrangement in the context of cell motility. The Rho GTPases directly linked to cytoskeletal reorganization in the context of cancer cell invasion and migration is the two Rho isoforms RhoA and RhoC. Literature evidence exists that these two isoforms of Rho GTPases may impart opposing effects on cancer metastasis, yet detailed analysis of signaling pathways that could contribute to such process has been acutely lacking. In this work, we elucidate the mechanism by which RhoC imparts highly specific downstream signaling effects different than RhoA and in separate cellular compartments at the leading edge of cell protrusions during EGF-stimulated motility. We will address this problem by directly visualizing multiple protein activities simultaneously in living cells, using novel biosensors that are proposed here. Aim1: Visualize two protein activities simultaneously in single living cell and in real-time using fully genetically encoded approach. Aim2: Develop new biosensors for downstream Rho effectors ROCK-1 and mDia1, amenable to simultaneous visualization together with the specific upstream Rho isoform Aim3: Investigate the spatiotemporal segregation of signaling coordinating RhoC and its downstream effector pathways at the leading edge Aim4: Investigate the spatiotemporal signaling coordinating RhoA and its downstream effector pathways at the leading edge These studies will produce new technologies valuable in direct visualization of Rho GTPase isoforms and their immediate downstream effector activations, enabling further spatiotemporal delineation of signaling mechanisms. Through these studies, we will be able to dissect the mechanism of the leading edge protrusions controlled through differential activities of Rho isoforms in breast carcinomas and enable us to address the specific role RhoC plays in producing the localized and polarized protrusions at the leading edge of breast carcinomas. PUBLIC HEALTH RELEVANCE: Rho-subfamily of p21 small GTPases has been postulated to exhibit a complex coordination of their activities in space and time, depending on particular environmental cues stimulating cell motility. However, high-resolution imaging studies of these coordinated balance of GTPase activities have been acutely lacking, due primarily to the technical challenges of imaging multiple protein activities in living cells. Rho-GTPases directly linked to cytoskeletal reorganization in the context of cancer cell invasion and migration, are the two Rho isoforms RhoA and RhoC. Here, we visualize simultaneously these Rho isoform activities together with activations of their downstream effectors in a single living breast carcinoma cell and address signaling events that coordinate the leading edge protrusions in response to EGF stimulation. Our key hypothesis is that RhoC activation in breast carcinomas in response to growth factor stimulation could impart a subcellular signal polarization mechanism that promotes effective forward protrusion of the leading edge through segregation of highly active versus complete inhibitive zones of actin polymerization within the leading edge.